Techniques for thermal cycling of DNA samples are known in the art. By performing a polymerase chain reaction (PCR), DNA can be amplified. It is desirable to cycle a specially constituted liquid biological reaction mixture through a specific duration and range of temperatures in order to successfully amplify the DNA in the liquid reaction mixture. Thermocycling is the process of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of double stranded DNA. The liquid reaction mixture is repeatedly put through this process of melting at high temperatures and annealing and extending at lower temperatures.
In a typical thermocycling apparatus, a biological reaction mixture including DNA will be provided in a large number of sample wells on a thermal block assembly. Quantitative PCR (qPCR) uses fluorogenic probes to sense DNA. Instrumentation designed for qPCR must be able to detect approximately 1 nM of these probes in small volume samples (e.g., approximately 25 μl). The detection method must be compatible with the thermal cycling required for qPCR. The detection method must also be capable of distinguishing multiple fluorogenic probes in the same sample.
Enhancing the sensitivity of fluorescence detection of a qPCR instrument or method improves the usefulness of that instrument or method by enabling detection of DNA sooner, that is, after fewer thermal cycles.
Prior art systems use the same light path for excitation and detection. In those systems excitation light is directed to a beam splitter, which transmits typically about one-half of the excitation light to the sample. Some of the emitted light from the sample comes back, to the beam splitter and a portion of that light, typically about one-half, is directed to a detector. By using beam splitters, only about one-half of the light is reflected and transmitted; therefore, only about one-quarter of the signal is measured.
U.S. Pat. No. 5,757,014 to Bruno et al. discloses an optical detection device for analytical measurements of chemical substances. The Bruno et al. device includes an excitation light guide and an emission light guide that share the same optical light path. U.S. Pat. No. 6,563,581 to Oldham et al. discloses a system for detecting fluorescence emitted from a plurality of samples in a sample tray. The Oldham et al. device includes a plurality of lenses, an actuator, a light source, a light direction mechanism and an optical detection system. U.S. Pat. No. 6,015,674 to Woudenberg et al. discloses a system for measuring in real time polynucleotide products from nucleic acid amplification processes, such as polymerase chain reaction (PCR). The Woudenberg et al. device includes a sample holder, an optical interface, a lens, and a fiber optic cable for delivering an excitation beam to a sample and for receiving light emitted by the sample.
Other prior art methods use fiber optics to deliver the excitation light to and collect the fluorescence from the sample. These methods may either use independent fiber optics for each sample or scan the same fiber optics over all the samples. Some methods illuminate the entire collection of samples simultaneously and detect the fluorescence with large area detectors.